• Journal of Microbiology
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• v.39 no.1
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• pp.62-66
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• 2001

Unlike eukaryotic efflux pumps energized by ATPase bacterial efflux pumps are energized by the proton motive force. That is the reason why CCCP, an inhibitor of proton motive forcer is widely used to study the bacterial efflux pump. In many cases, efflux systems have been observed only in quinolone-resistant bacteria. Most of the quinolone-susceptible strains have been found to maintain little efflux pump. However some susceptible bacteria skewed the increased intracellular quinolone concentration only at a low concentration (0.01 or 0.1 mM) but net at a high concentration (1 mM) of CCCP. If bacterial cells were killed at high concentrations of CCCP and lost the integrity of their membranes, the intracellular quinolone would leak out from cells with no efflux system. The efflux pump system in the quinolone-susceptible strains could net be detected at the same concentration used for resistant bacteria. To test this hypothesist the intracellular quinolone concentration in the quinolone-susceptible and -resistant strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was assayed at various concentrations of CCCP. Since the effect of CCCP is very rapid, the survival of bacteria was observed by assaying the DNA synthesis in 5 min. In the case of E. coli, but not P. aeruginosa or S. aureus, the quinolone susceptible strain was more susceptible to CCCP than the quinolone resistant ones, especially when the incubation with CCCP was extended. Decrease of the intracellular quinolone concentration resulted in a false result-no or weak efflux system in the quinolone susceptible strains. Results suggested that the concentration of CCCP should be optimized in order to detect the efflux system in the quinolone susceptible strains of E. coli.

• YAKHAK HOEJI
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• v.60 no.1
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• pp.1-7
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• 2016

The aim of this study was to investigate the prevalence of quinolone resistant E. coli from retail meat and to characterize the resistant determinants. Determination of minimum inhibitory concentration, the sequence analysis of gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR), the presences of plasmid mediated quinolone resistance (PMQR) and the expression of efflux pump genes were investigated. Of the total 277 retail meat samples, 67 coli form bacteria were isolated. 15 of 67 isolates showed nalidixic acid resistance and 7 of 15 nalidixic acid resistant isolates were also resistant to ciprofloxacin, moxifloxacin and levofloxacin. 11 of 15 nalidixic acid resistant strains were isolated from chicken, 2 of 15 were isolated from beef and 2 of 15 were isolated from pork samples. 11 of 15 nalidixic acid resistant strains have single mutation at codon 87 (D87N or D87G) in gyrA, 2 of 11 gyrA mutants have double mutations at codon 86 and 87 (L86A and L87I) in parC with mutations at codon 434+445+465 or 429 in gyrB. 2 of 15 resistant isolates harbored qnrS, a PMQR determinant. Over expression of the acrB gene, efflux pump gene (3.93~16.53 fold), was observed in 10 of 15 resistant isolates.

• Korean Journal of Microbiology
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• v.50 no.3
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• pp.185-190
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• 2014

The aim of this study was to investigate the prevalence of quinolone resistant E. coli from raw bulk milk and to characterize the resistance determinants. In this study, the gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced from quinolone resistant E. coli isolates. Also, the presence of plasmid-mediated quinolone resistance (PMQR) and the expression of efflux pump genes based on quantitative real-time PCR (qRT-PCR) were investigated. Of the 487 coliform bacteria, 9 strains showed nalidixic acid resistance, and 6 of the 9 nalidixic acid resistant isolates were also ciprofloxacin resistant. These 9 strains had a single mutation at codon 83 (S83L) in gyrA, 2 of them had double mutations at codon 83 and 87 (S83L and D87N) in gyrA and 3 of the 9 isolates had single mutations at codon 80 (S80I) in parC. None of the 9 isolates harbored PMQR determinants. Compared with wild-type E. coli ATCC 25922, an over-expression of the acrB gene (2.15-5.74 fold), encoding the pump component of the AcrAB-TolC efflux pump was observed in 4 of 6 ciprofloxacin resistant isolates. This study identified the quinolone resistance mechanism of E. coli isolated from raw milk samples in Gyeonggi-do.

• Microbiology and Biotechnology Letters
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• v.52 no.1
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• pp.99-101
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• 2024

Fluoroquinolones have been extensively used in treatment or prevention of serious infections in humans and food-producing animals, especially in poultry, due to their broad-spectrum activity to both Gram-positive and -negative pathogens. However, the use of fluoroquinolones in poultry production selects for bacteria carrying genetic determinants for quinolone resistance. Here, we present the complete genome sequences of two methicillin-resistant Staphylococcus aureus (MRSA) strains displaying quinolone resistance, which were isolated from healthy broilers in Korea.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.4
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• pp.422-430
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• 2018

The inappropriate and widespread use of quinolones in humans and animals may cause accelerated emergence and spread of antimicrobial-resistant determinants. In this study, we investigated quinolone resistance mechanisms in a total of 65 nalidixic acid-resistant E. coli isolated from swine rectal swabs (N=40) and clinical specimens (N=25). Antimicrobial susceptibilities were determined by the disk diffusion method. PCR and DNA sequencing were performed for investigations of genes and mutations associated with quinolone resistance. In our study, 62 of 65 nalidixic acid-resistant E. coli harbored mutations in gyrA, parC, and/or parE genes; of the 65 isolates, 62 (95.4%) had mutations in the gyrA gene, 35 (53.8%) had mutations in the parC gene, 7 (10.8%) had mutations in the parE gene. The 35 isolates harbored mutations in two genes, gyrA and parC. Plasmid-mediated quinolone resistance (PMQR) determinants were investigated in the 65 isolates. Thirteen of 65 nalidixic acid-resistant E. coli contained the qnrS gene and 10 of those isolates had mutations in the gyrA, parC, and/or parE genes. We confirmed that an important mechanism of quinolone resistance in E. coli isolated from human and swine involves chromosomal mutations in the gyrA, parC, and/or parE genes with increasing use of quinolone for treatment or additives.

• Journal of Microbiology
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• v.45 no.6
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• pp.590-592
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• 2007

We report the isolation of Salmonella enterica serotype Typhimurium phage type DT104 (CCARM 8104) from swine in Korea. The CCARM 8104 isolate was resistant to nalidixic acid and showed reduced susceptibility to quinolones. The CCARM 8104 isolate had a missense mutation, Asp87Asn, in the quinolone resistance-determining region in gyrA and produced PSE-1. The CCARM 8104 isolate carried two different class 1 integrons, and the PSE-1 ${\beta}$-lactamase gene was inserted into a 1,200 bp class 1 integron. The presence of DT104 with pse-1 in an integron located in a plasmid and reduced susceptibility to quinolone in swine pose a significant threat of possible horizontal spread between swine and humans.

• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1180-1183
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• 2020

The prevalence and characterization of plasmid-mediated quinolone resistance (PMQR) determinants in ciprofloxacin-resistant Escherichia coli isolated from a Korean commercial layer farm were studied. A total of 45 ciprofloxacin-resistant E. coli isolates were recovered and all isolates were multidrug-resistant. Eight isolates have the PMQR genes aac(6')-Ib-cr, qnrS1, and qnrB4, and seven isolates exhibited double amino acid exchange at both gyrA and parC, and have high fluoroquinolone minimum inhibitory concentrations. Five transconjugants demonstrated transferability of PMQR and β-lactamase genes and similar antimicrobial resistance. Because PMQR genes in isolates from commercial layer chickens could enter the food supply and directly affect humans, control of ciprofloxacin resistance is needed.

• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1853-1857
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• 2008

Salmonella contamination in chicken meat was studied with 100 chicken meat samples purchased from 55 shops located in various regions. A total of 21 isolates of Salmonella enterica were isolated from 21 chicken meat samples from four shops located at open markets, whereas there were none from supermarkets with well-equipped cold systems. Among these, 18 isolates were identified as Salmonella enterica serotype Haardt (S. Haardt) and three isolates were S. enterica serotype Muenchen. When the minimal inhibitory concentrations of the S. Haardt isolates were assayed with the agar dilution method to determine susceptibility to ampicillin, chloramphenicol, sulfisoxazole, tetracycline, and nalidixic acid, all 18 isolates were resistant to tetracycline and nalidixic acid and nine of these were resistant to ampicillin. These isolates showed reduced susceptibility to eight fluoroquinolones including ciprofloxacin, enrofloxacin, levofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, norfloxacin, and ofloxacin. When quinolone resistance determining regions of gyrA and gyrB were sequenced, every isolate had the same missense mutation Ser83$\rightarrow$Tyr (TCC$\rightarrow$+TAC) in gyrA, whereas no mutation was found in gyrB. Pulsed-field gel electrophoresis with XbaI revealed a close relationship among these isolates, suggesting a contamination of raw chicken meat with clonal spread of nalidixic acid-resistant and quinolone-reduced susceptibility S. Haardt in chickens. Results in this study show the importance of a well-equipped cold system and the prudent use of fluoroquinolone in chickens to prevent the occurrence of quinolone-resistant isolates.

• Biomedical Science Letters
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• v.26 no.2
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• pp.120-126
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• 2020

Fluoroquinolone (FQ) resistant uropathogenic Escherichia coli (UPEC) have become a major problem in urinary tract infections (UTIs). The purpose of this study was to compare the quinolone resistance-determining region (QRDR) and plasmid mediated quinolone resistance (PMQR) determinants of FQ resistant UPEC between 1989 and 2010-2014. A total of 681 strains of UPEC clinical isolates was collected from Korean healthcare facility in 1989 (123 strains) and in 2010-2014 (558 strains). The minimum inhibitory concentrations (MICs) of FQs were determined by agar dilution method. QRDRs (gyrA, gyrB, parC and parE) and PMQR determinants (qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA) were analyzed polymerase chain reaction and sequencing method. Among 681 isolates, FQ resistant UPEC were 3 strains (2.4%) in 1989 isolates and 220 strains (39.4%) in 2010-2014 isolates. The rate of the FQ resistant UPEC strains in 2010-2014 isolates was increased than that of in 1989 isolates. UPEC isolates from 1989 and 2010-2014 were shown to carry mutations in gyrA (Ser83 and Asp87), gyrB (Ser464 and Thr469), parC (Ser80 and Glu84) and parE (Glu460, Ser458, Ile464 and Leu445). The most common mutations of QRDRs in 1989 isolates were Ser83Leu and Asp87Gly in gyrA and Ser80Ile in parC (2 strains: 66.7%) while those in 2010-2014 isolates were Ser83Leu and Asp87Asn in gyrA and Ser80Il2 and Glu84Val in parC (88 strains: 40.0%). PMQR determinants were detected only in 2010-2014 UPEC strains (47 strains: 21.4%).

• Archives of Pharmacal Research
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• v.19 no.5
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• pp.353-358
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• 1996

E. coli 11 and E. coli 101, clinical isolates of Escherichia coli were resistant to various quinolones, especially MICs to norfloxacin of both strains were higher than 100 mg/ml. In the presence of carbonyl cyanide m-chlorophenylhydrazone, a proton gradient uncoupler, norfloxacin uptake in both strains was increased, suggesting that an efflux system play an important role in the norfloxacin resistance. Outer membrane proteins of the susceptible and resistant strains which could affect the route of norfloxacin entry into cells were different. When quinolone resistance determining region(QRDR) of gyrA was amplified using PCR and cut with Hinf I, QRDR in the susceptible strain yielded two fragments while QRDRs in E. coli 11 and E. coli 101 yielded only one uncut fragment. When DNA sequence of QRDR was analyzed, there were two mutations as Ser-83 and Asp-87 in both resistant strains. these residues were changed to Leu-83 and Asn-87, respectively. These results showed that the norfloxacin resistance of E. coli 11 and E. coli 101 was resulted from multiple changes-an altered DNA gyrase A subunit, a change in route of drug entry, and reduction in quinolone concentration inside cells due to an efflux system.